Differentiation is the consequence of two steps, an exit from pluripotency and an entrance into a specific lineage. Cells are known to be primed for differentiation based on cell-to-cell variation in gene expression, or cellular heterogeneity, although the mechanism remains unknown. Fundamentally, gene expression is regulated by nuclear architecture, defined as the three dimensional, temporally-regulated architecture of chromatin. This study focuses on the role of nuclear architecture in setting the foundation for cellular heterogeneity to permit the exit of a stem cell from pluripotency and entrance into hematopoiesis. Two key contributors to nuclear architecture are enhancers and CCCTC-binding factor (CTCF) binding sites. Enhancers are cis-regulatory elements that play a critical role in regulating lineage-specific transcription. CTCF binds sites throughout the genome and regulates gene expression by permitting or preventing enhancer- promoter interactions. Neither regulatory element has been studied as a modulator of cellular heterogeneity. This study will show that the nuclear architecture of a cell is vital to directing stem cell differentiation into hematopoietic lineages through regulation of cellular heterogeneity. This proposal focuses on how stem cells exit pluripotency through changes in Nanog expression (Aim 1), and successively enter hematopoietic lineages by observing changes in the hematopoietic-essential HoxA cluster (Aim 2). For Aim 1, the role of nuclear architecture in heterogeneity will be examined by single-cell RT- qPCR through the CRISPR/Cas9 mediated deletion in murine embryonic stem cells (ESCs) of 1) the three Nanog-associated enhancers and 2) the CTCF sites bounding Nanog. For Aim 2, CTCF sites within the HoxA cluster will be deleted in ESCs. These cells will then be differentiated to hematopoietic lineages and changes in heterogeneity evaluated using single-cell RT-qPCR. In both aims, changes in enhancer-promoter interactions will be investigated using Chromosome Conformation Capture (3C). In sum, although cellular heterogeneity is critical to stem cell differentiation and lineage-specification, its mechanism remains unknown. This study will establish the paradigm that nuclear architecture modulations are responsible for cellular heterogeneity and driving differentiation. Better understanding of the regulation of stem cell differentiation and hematopoiesis will further our ability to understand how these processes are perturbed in disease and development. Importantly, as the role of personalized medicine and stem cells as therapies grows in the clinical arena, the understanding of the nuances of stem cell differentiation and biology this fellowship will provide the applicant is a rigorous training ground for her future as an independent physician- scientist. The guidance in the vibrant and intellectual environment, as well as the clinical shadowing, conference attendance and coursework described in this fellowship, will position the applicant to be a competitive candidate for research-based residency programs.